Exposure of PC12 cells to ethanol results in large changes in calcium-stimulated protease activities. Since these proteases are critical modulators of both neurotransmitter release as well as cellular toxicity and death, we are exploring the hypothesis that ethanol-mediated neural toxicity may result from calcium- activated protease dysregulation. Calpastatin, an inhibitor of calpain, is an acidic, hydrophobic protein which interacts with the hydrophobic active site(s) of mu- and m-calpains. A series of post-translational modifications of calpastatin have been described which alter the binding affinity to calpains, among them, PKC-mediated phosphorylations. Using a PC12 model, we are examining the effects of ethanol exposure and withdrawal on protein-protein interactions, and will determine what, if any, post-translational modifications occur to alter these interactions. A variety of techniques are used for these studies, including gel filtration, fluorescence-based protease assays, identification of protein-protein complexes using a variety of immunochemical techniques and mass-mapping of isolated protein complexes. Because of the hydrophobic nature of calpastatin-calpain interactions, the possibility that ethanol might modify protease-inhibitor complex stability is being entertained.